Analysis of porcine macrophage immune response to antigenic molecules and short chain fatty acids / 동물의과학연구지

Macrophages play an important role in both the innate and adaptive immune responses. These include phagocytosis, killing of microorganisms, antigen presentation, and induction of immune cytokines and antimicrobial genes. Macrophage activity is reported to be controlled by diverse exogenous antigenic or endogenous metabolic molecules, and the underlying mechanisms are well documented in human and mouse macrophage cells. Bacterial lipopolysaccharide (LPS) is known to be one of the most potent stimuli activating macrophages through the toll like receptor 4 (TLR4) signaling pathway. There are other antigenic molecules, such as muramyl dipeptide (MDP) and outer membrane protein A (OmpA), that are also known to activate immune cells. On the other hand, short chain fatty acids (SCFAs) such as acetate and butyrate are produced by gut microbiota and control host energy metabolism and signal transduction through GPR receptors. However, there are few studies demonstrating the effects of these molecules in macrophages from domestic animals, including domestic pigs. In this study, we attempted to characterize gene expression regulation in porcine macrophages (PoM2, Pig Monocytes clone 2) following treatment with LPS, MDP, OmpA, and two short chain fatty acids using porcine genome microarray and RT-PCR techniques. A number of novel porcine genes, including anti-microbial peptides and others, appeared to be regulated at the transcriptional level. Our study reports novel biomarkers such as SLC37A2, TMEN184C, and LEAP2 that are involved in the porcine immune response to bacterial antigen LPS and two short chain fatty acids.

Activation of NOD2 signalling pathway stimulates the function of human dendritic cells loaded with leukemia cell lysates / 中国实验血液学杂志

The purpose of this study was to explore the effect of NOD2 signalling pathway activated by muramyl dipeptide (MDP) on the immunomodulation effect of human monocyte-derived dendritic cells (DC) loaded with leukemia cell lysates. Peripheral blood mononuclear cells (PBMNC) were isolated by density gradient centrifugation, These cells were cultured with three cytokines for 7 days to induce their maturation. On the 5th day, cells were loaded with leukemia cell HL-60 lysates. NOD2 expression was detected by RT-PCR and Western blot. The phenotype of DC were analyzed by flow cytometry, and ELISA was used to assay levels of IL-12 (p40) . The results showed that MDP could trigger NOD2 mRNA and protein expression in different groups of DC, especially in sensitized DC+MDP group, which was significantly higher than that in the DC+MDP group and sensitized DC without MDP stimulation, the difference was statistically significant (P < 0.05). Besides, the expression of surface molecules (HLA-DR, CD80, CD83, CD86, CD40) in the group of DC loaded with leukemia cell lysate and stimulated by MDP (sensitized DC+MDP) reached the highest level, followed by the group of DC loaded with leukemia cell lysate without MDP and DC only stimulated by MDP, non-treated DC were the lowest (P < 0.05). Similarly, compared with untreated unstimulated DC, after loading with HL-60 lysates or only stimulating with MDP, the secretion of IL-12p40 increased, but IL-12p40 level (573.86 ± 32.09 pg/ml) in DC+MDP group was higher than that in group of sensitized DC (365.03 ± 28.86 pg/ml) (P < 0.05), and it in sensitized DC+MDP group reached the highest (898.30 ± 61.08) pg/ml, compared to other groups (P < 0.05). It is concluded that MDP can significantly enhance the NOD2 mRNA and protein expression in sensitized DC, promote the expression of HLA-DR, synergistic costimulatory molecules and adhesion molecules of DC, at the same time, MDP can increase secretion of inflammatory factors IL-12p40. This study will provide a new ideas for DC application in leukemia immunotherapy.

Effect of neurotrophic factor, MDP, on rats’ nerve regeneration

Our objective was to determine the immune-modulating effects of the neurotrophic factor N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) on median nerve regeneration in rats. We used male Wistar rats (120-140 days of age, weighing 250-332 g) and compared the results of three different techniques of nerve repair: 1) epineural neurorrhaphy using sutures alone (group S - 10 rats), 2) epineural neurorrhaphy using sutures plus fibrin tissue adhesive (FTA; group SF - 20 rats), and 3) sutures plus FTA, with MDP added to the FTA (group SFM - 20 rats). Functional assessments using the grasp test were performed weekly for 12 weeks to identify recovery of flexor muscle function in the fingers secondary to median nerve regeneration. Histological analysis was also utilized. The total number and diameter of myelinated fibers were determined in each proximal and distal nerve segment. Two indices, reported as percentage, were calculated from these parameters, namely, the regeneration index and the diameter change index. By the 8th week, superiority of group SFM over group S became apparent in the grasping test (P = 0.005). By the 12th week, rats that had received MDP were superior in the grasping test compared to both group S (P < 0.001) and group SF (P = 0.001). Moreover, group SF was better in the grasping test than group S (P = 0.014). However, no significant differences between groups were identified by histological analysis. In the present study, rats that had received MDP obtained better function, in the absence of any significant histological differences.

Effects of muramyl dipeptide on the differentiation of human dental pulp cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the nucleotide-binding oligomerization domain-2 (NOD-2) gene expression in deep caries and the effects of NOD-2 agonist muramyl dipeptide (MDP) on the differentiation of human dental pulp cells (hDPC).</p><p><b>METHODS</b>NOD-2 gene level in deep caries and healthy pulp tissue was determined by real-time quantitative polymerase chain reaction (realtime-PCR). Realtime-PCR, Western blotting and immunofluorescence were performed to evaluate NOD-2 gene and protein expression. Dentin sialoprotein (DSP) protein level was assessed when hDPC were challenged by different concentrations of MDP for 24 hours, and sialophosphoprotein (DSPP), osteocalcin (OCN) mRNA and osteopontin (OPN) protein level were detected at different time points after incubation with 0.1 mg/L MDP.</p><p><b>RESULTS</b>NOD-2 mRNA level was higher in pulp tissue of deep caries (0.2610 ± 0.0824) than that in healthy controls (0.0024 ± 0.0002), P < 0.05. The expression of NOD-2 gene and protein increased in a time denpendent manner upon stimulation with MDP. Immunofluorescence confirmed that NOD-2 protein was located in cytoplasm. Moreover, 0.1 mg/L MDP augmented DSP protein level. DSPP and OCN mRNA were elevated with time and reached the peak at 12 h and down-regulated. OPN protein level also increased with time.</p><p><b>CONCLUSIONS</b>Dental pulp NOD-2 expression are up-regulated in pulp tissue of deep caries. MDP may be related to the differentiation of hDPC.</p>

Study on the hypochlolesterolemic and antioxidative effects of tyramine derivatives from the root bark of Lycium chenese Miller

The aim of the present study was to investigate the hypocholesterolemic effect and potential of tyramine derivatives from Lycii Cortex Radicis (LCR), the root bark of lycium (Lycium chenese Miller) in reducing lipid peroxidation. The activities of enzymes, hepatic 3-hydroxy 3-methylglutaryl (HMG) CoA reductase and acyl-CoA:cholesterol acyltransferase (ACAT) and LDL oxidation were measured in vitro and animal experiments were also performed by feeding LCR extracts to rats. The test compounds employed for in vitro study were trans-N-p-coumaroyltyramine (CT) and trans-N-feruloyltyramine (FT), LCR components, N-(p-coumaroyl)serotonin (CS) and N-feruloylserotonin (FS) from safflower seeds, ferulic acid (FA) and 10-gingerol. It was observed that FT and FS at the concentration of 1.2 mg/mL inhibited liver microsomal HMG CoA reductase activity by ~40%, but no inhibition of activity was seen in the cases of CT, CS, FA and 10-gingerol. Whereas, ACAT activity was inhibited ~50% by FT and CT, 34-43% by FS and CS and ~80% by 10-gingerol at the concentration of 1 mg/mL. A significant delay in LDL oxidation was induced by CT, FT, and 10-gingerol. For the animal experiment, five groups of Sprague-Dawley male rats were fed high fat diets containing no test material (HF-control), 1 and 2% of LCR ethanol extract (LCR1 and LCR2), and 1% of extracts from safflower seed (Saf) and ginger (Gin). The results indicated that total cholesterol level was significantly lower in Saf, LCR2 and Gin groups, and HDL cholesterol level was lower only in Gin group when compared with HF-control group; while there was no difference in the serum triglyceride levels among the five experimental groups. The level of liver cholesterol was significantly lower in LCR1 and LCR2 groups than HF-control. Serum levels of TBARS were significantly lower only in LCR2 group when compared with HF-control group. From the observed results, we concluded that LCR can be utilized as a hypocholesterolemic ingredient in combination with ginger, especially for functional foods.

A Case of Superficial Acral Fibromyxoma / 대한피부과학회지

Superficial acral fibromyxoma (SAF) is an uncommon soft tissue tumor that has recently been showed to be a separate disease entity. It is most often located in the ungal region of the fingers and toes of middle-aged adults. It is histologically characterized by a slight to moderate cellular proliferation of spindle cells and stellate cells arranged in a random, storiform or fascicular pattern, within a myxoid, myxocollagenous or collagenous stroma with a prominent vasculature. The neoplastic cells show positive staining for CD34, CD99 and EMA, but negative staining for S100, HMB45, cytokeratin, smooth muscle actin (SMA) and desmin. We report here on a typical case of SAF that occurred on the left index finger of a 44-year-old man.

Effect of muramyl dipeptide on proliferation of dendritic cells derived from children acute leukemia bone marrow in vitro / 中国实验血液学杂志

The aim of this study was to explore the effect of muramyl dipeptide (MDP) on proliferation of dendritic cells (DCs) from bone marrow of children with acute leukemia in vitro. The mononuclear cells were isolated from bone marrow of children with acute leukemia to induce dendritic cells. The experiment was divided into 4 groups. The control group: MNC + RPMI 1640 medium; test group 1: MNC + MDP; test group 2: MNC + rhGM-CSF + IL-4 + rhTNFα; test group 3: MNC + rhGM-CSF + IL-4 + rhTNFα + MDP. The growth of DCs was observed by inverted microscope every day; the number of DCs in different groups were counted, the immunophenotypes of DCs were detected by flow cytometry on day 8 of culture. The results indicated that a certain number of typical DCs could be detected in all experimental groups. The DC number in control and 3 test groups were (0.85 ± 0.23) x 10⁵/L, (2.31 ± 0.24) x 10⁵/L, (3.26 ± 0.37) x 10⁵/L and (4.16 ± 0.34) x 10⁵/L, respectively, among which DC number is in all 3 test groups were higher than that in control group (p < 0.01), the DC number in test group 1 was lower than that in test groups 2 and 3 (p < 0.01), while it in test group 3 was higher than that in test group 2 (p < 0.01). The percentages of HLA-DR in control, test group 1, 2 and 3 were 19.98 ± 3.74, 37.24 ± 4.32, 58.81 ± 2.08 and 77.48 ± 5.57 respectively; the percentages of CD1a and CD83 in control, test group 1, 2 and 3 were 11.46 ± 2.43, 28.71 ± 6.64, 46.92 ± 4.78 and 57.03 ± 3.07, as well as 13.05 ± 5.70, 36.32 ± 5.61, 54.95 ± 7.83 and 75.70 ± 6.67 respectively. The comparison of HLA-DR, CD1a and CD83 levels in control and test group 1, 2 showed that their results were consistent with results of DC numbers. It is concluded that MDP not only promotes the proliferation of DCs derived from bone marrow of children with acute leukemia in vitro, cooperates with rhGM-CSF, rhIL-4 and rhTNFalpha in promoting of the proliferation and maturation of DCs, while the promotive effect of MDP alone on the proliferation of DCs is not as good as its combination with cytokines.

Liposomal delivery of Mycobacterium leprae antigen(s) with murabutide and Trat peptide inhibits Fas-mediated apoptosis of peripheral blood mononuclear cells derived from leprosy patients.

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose capped lipoarabinomannan) may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with particulate mode of antigen presentation led to both decreased percentage of propidium iodide (PI) positive cells and T cells expressing Fas-FasL, as well as decreased caspase-8/-3 activities in the lepromatous patients, thereby inhibiting apoptosis, while converse was true with stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-X(L) in the lepromatous patients, thereby inhibiting apoptosis. Thus, the liposomal formulation of antigen promoted proliferation of anergized T cell by inhibiting apoptosis through decreased expression of death receptors and caspase activities and increased expression of anti-apoptotic protein Bcl-X(L) in these patients.

A Current Research Insight into Function and Development of Adjuvants

In recent years, adjuvants have received much attention because of the development of purified subunit and synthetic vaccines which are poor immunogens and require adjuvants to evoke the immune response. Therefore, immunologic adjuvants have been developed and testing for most of this century. During the last years much progress has been made on development, isolation and chemical synthesis of alternative adjuvants such as derivatives of muramyl dipeptide, monophosphoryl lipid A, liposomes, QS-21, MF-59 and immunostimulating complexes (ISCOMS). Biodegradable polymer microspheres are being evaluated for targeting antigens on mucosal surfaces and for controlled release of vaccines with an aim to reduce the number of doses required for primary immunization. The most common adjuvants for human use today are aluminum hydroxide and aluminum phosphate. Calcium phosphate and oil emulsions have been also used in human vaccination. The biggest issue with the use of adjuvants for human vaccines is the toxicity and adverse side effects of most of the adjuvant formulations. Other problems with the development of adjuvants include restricted adjuvanticity of certain formulations to a few antigens, use of aluminum adjuvants as reference adjuvant preparations under suboptimal conditions, non-availability of reliable animal models, use of non-standard assays and biological differences between animal models and humans leading to the failure of promising formulations to show adjuvanticity in clinical trials. The availability of hundreds of different adjuvants has prompted a need for identifying rational standards for selection of adjuvant formulations based on safety and sound immunological principles for human vaccines. The aim of the present review is to put the recent findings into a broader perspective to facilitate the application of these adjuvants in general and experimental vaccinology.

Effects of immunization with sperm specific lactate dehydrogenase with & without muramyl dipeptide as adjuvant.

The effect of sperm specific lactate dehydrogenase-C4 (LDH-C4) alone or with muramyl dipeptide (MDP) and Freund's complete adjuvant (FCA) on immune responses and breeding capacity have been studied in isogeneic C57 BI/Ks (H-2d) mice. Results per se suggested that LDH-C4 generates isoantibodies even in absence of adjuvant. Though MDP could be substituted for FCA as adjuvant, amplification by MDP of the antibody levels is reduced to half that obtained with FCA. LDH-B4 from kidney did not produce any antibody response under similar conditions. Systemic immunization with LDH-C4 did not reduce the overall pregnancy rate but reduced the frequency of embryo resorption and increased litter size significantly. However, mothers with foetal non-resorption had in general, lower antibody titres than the mothers showing foetal resorptions. The popliteal lymph node (PLN) assay for local graft versus host (LGVH) reaction revealed that the maternal lymphocyte cell competence was depressed by the sperm specific isozyme during gestation; depression in stimulation index was associated with the low antibody titre group. MDP, like LDH-C4 alone did not modify LGVH reaction significantly although it exerted a similar effect as LDH-C4 in embryo protection.

Effect of cisplatin, rIFN-Y, LPS and MDP on release of H2O2, O2- and lysozyme from human monocytes in vitro.

Increased secretion of H2O2, O2- and lysozyme by human monocytes in vitro on treatment with cisplatin, rIFN-Y (interferon-Y), LPS (lipopolysaccharide) and MDP (muramyl dipeptide) is reported. It is suggested that increased production of these secretory products represent the activated state of monocytes. These in vitro activated monocytes could either kill the tumor cells via increased contact mediated cytolysis or cytolysis mediated via the release of the secretory products like H2O2, O2- and lysozyme.

Adjuvant activity of some nonpyrogenic hydrophobic analogues of muramyl dipeptide in enhancing the primary humoral and cell mediated immune responses in guinea pig model.

Five muramyl dipeptide analogues synthesized by derivatization of gamma-carboxyl of D-isoglutamine residue of MDP into alkyl amides or incorporation of lysine residue at the site via epsilon-NH2 function were evaluated for immuno-adjuvant activity. Derivatization of gamma-carboxyl of D-isoglutamine into butyl, octyl and dibutyl residues stimulated delayed type of hypersensitivity (DTH) response, the maximum stimulation being observed with octyl amide. Introduction of lauryl amide residue abolished DTH response. The antibody response was impaired with all the alkyl amide analogues except for the lysyl amide derivative with which the response was higher than MDP. Correlation was observed between DTH response and macrophage migration.

Anti-stress activity in a muramyl-dipeptide.

N-Acetylmuramyl-L-alanyl-D-isoglutamine and some of its derivatives have been examined for anti-stress activity. Amongst these, N-palmitoylmuramyl-L-alanyl-D-isoglutamine is shown to considerably enhance the capacity of animals to endure various types of stress. This indicates that besides acting as immunomodulators and sleep regulators, muramyl dipeptides may also act as anti-stress agents.

Muramil dipeptido (MDP): origen, mecanismo de acción y perspectivas / Muramyl dipeptide: origin, mechanism of action and perspectives

En el presente trabajo se hace una revisión de los aspectos teóricos y experimentales del componente mínimo que reproduce el efecto inmunorregulador del adyuvante completo de Freund: el muramil dipéptido (MDP). Se hace un breve análisis de su historia y de las principales evidencias experimentales que permiten proponer el mecanismo de acción del MDP sobre células inmunocompetentes, además de la importancia que esta molécula tiene como inmunorregulador y las perspectivas de su uso